The present invention relates to variants of microbial cell-wall degrading enzymes, more specifically to variants of enzymes having a pectinase structure similar to that of Bacillus licheniformis enzymes exhibiting pectate lyase activity as their major enzymatic activity in the neutral and alkaline pH ranges; to a method of producing such enzymes; and to methods for using such enzymes in the textile, detergent and cellulose fiber processing industries. The enzyme variants of the invention may exhibit increased thermostability as compared to the parent enzyme.
Plant cell walls consist of a complicated network of fibrous materials. The composition of the cell walls varies considerably, depending on the source of the vegetable material. However, in general its composition can be summarized as mainly comprising non-starch polysaccharides. These polysaccharides can be found in various forms: cellulose, hemicellulose and pectins.
The composition of a plant cell wall is both complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising e.g. various .beta.-xylan chains) and pectin. The occurrence, distribution and structural features of plant cell wall polysaccharides are determined by: 1. Plant species; 2. Variety; 3. Tissue type; 4. Growth conditions; and 5. Ageing (Chesson (1987), Recent Advances in Animal Food Nutrition, Haresign on Cole, eds.). Butterworth, London, 71-89).
Basic differences exist between monocotyledons (e.g. cereals and grasses) and dicotyledons (e.g. clover, rapeseed and soybean) and between the seed and vegetative parts of the plant (Carre"" and Brillouet (1986), Science and Food Agric. 37, 341-351). Monocotyledons are characterized by the presence of an arabinoxylan complex as the major hemicellulose backbone. The main structure of hemicellulose in dicotyledons is a xyloglucan complex. Moreover, higher pectin concentrations are found in dicotyledons than in monocotyledons. Seeds are generally very high in pectic substances, but relatively low in cellulosic material. Three more or less interacting polysaccharide structures can be distinguished in the cell wall:
1. The middle lamella forms the exterior cell wall. It also serves as the point of attachment for the individual cells to one another within the plant tissue matrix. The middle lamella consists primarily of calcium salts of highly esterified pectins;
2. The primary wall is situated just inside the middle lamella. It is a well-organized structure of cellulose microfibrils embedded in an amorphous matrix of pectin, hemicellulose, phenolic esters and proteins;
3. The secondary wall is formed as the plant matures.
During the plant""s growth and ageing phase, cellulose microfibrils, hemicellulose and lignin are deposited.
There is a high degree of interaction between cellulose, hemicellulose and pectin in the cell wall. The enzymatic degradation of these rather intensively cross-linked polysaccharide structures is not a simple process. A large number of enzymes are known to be involved in the degradation of plant cell walls. They can broadly be subdivided in cellulases, hemicellulases and pectinases (Ward and Young (1989), CRC Critical Rev. in Biotech. 8, 237-274).
Cellulose is the major polysaccharide component of plant cell walls. It consists of .beta. 1,4 linked glucose polymers.
Cellulose can be broken down by cellulases, also called cellulolytic enzymes. Cellulolytic enzymes have been divided traditionally into three classes: endoglucanases, exoglucanases or cellobichydrolases and .beta.-glucosidases (Knowles, J., et al. (1987), TIBTECH 5, 255-261). Like all cell wall degrading enzymes they can be produced by a large number of bacteria, yeasts and fungi. Apart from cellulases degrading .beta.-1,4 glucose polymers, endo-1,3/1,4 beta.-glucanases and xyloglucanases should be mentioned (Ward and Young op. cit.).
Pectins are major constituents of the cell walls of edible parts of fruits and vegetables. The middle lamella which are situated between the cell walls are mainly built up from protopectin which is the insoluble form of pectin. Pectins are considered as intracellular adhesives and due to their colloidal nature they also have an important function in the water regulation system of plants. The amount of pectin can be very high. For example, lemon peels are reported to contain pectin at up to 30% of their dry weight, orange peels contain from 15-20% and apple peels about 10% (Norz, K. (1985). Zucker und Susswaren Wirtschaft 38, 5-6).
Pectins are composed of a rhamno-galacturonan backbone in which 1,4-linked (.alpha.-D-galacturonan chains are interrupted at intervals by the insertion of 1,2-linked (.alpha.-L-rhamnopyranosyl residues (Pilnik, W. and A. Voragen (1970), In: The Biochemistry of fruits and their products, vol. 1, Chapter 3, p. 53. Acad. Press). Other sugars, such as D-galactose, L-arabinose and D-xylose, are present as side chains. A large part of the galacturonan residues is esterified with methyl groups at the C2 and C3 position.
A large number of enzymes are known to degrade pectins. Examples of such enzymes are pectin esterase, pectin lyase (also called pectin transeliminase), pectate lyase, and endo- or exo-polygalacturonase (Pilnik and Voragen (1990). Food Biotech 4, 319-328). Apart from enzymes degrading smooth regions, enzymes degrading hairy regions such as rhamnogalacturonase and accesory enzymes have also been found (Schols et al. (1990), Carbohydrate Res. 206, 105-115; Searle Van Leeuwen et al. (1992). Appl. Microbiol. Biotechn. 38, 347-349).
Pectinases can be classified according to their preferential substrate, highly methyl-esterified pectin or low methyl-esterified pectin and polygalacturonic acid (pectate), and their reaction mechanism, beta-elimination or hydrolysis. Pectinases can be mainly endo-acting, cutting the polymer at random sites within the chain to give a mixture of oligomers, or they may be exo-acting, attacking from one end of the polymer and producing monomers or dimers. Several pectinase activities acting on the smooth regions of pectin are included in the classification of enzymes provided by the Enzyme Nomenclature (1992) such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67), exo-polygalacturonate lyase (EC 4.2.2.9) and exo-poly-alpha-galacturonosidase (EC 3.2.1.82).
Pectate lyases degrade un-methylated (polygalacturonate) or low-methylated pectin by xcex2-elimination of the xcex1-1,4-glycosidic bond. The enzymes are generally characterised by an alkaline pH optimum, an absolute requirement for Ca2+ (though its role in binding and catalysis is unknown) and good temperature stability.
Pectate lyases have been cloned from different bacterial genera such as Bacillus, Erwinia, Pseudomonas, Klebsiella and Xanthomonas.
U.S. Pat. No. 6,124,127, which is hereby incorporated by reference in its entirety, discloses a cloned Bacillus licheniformis pectate lyase. The DNA sequence encoding this B. licheniformis pectate lyase and the deduced amino acid sequence are listed in the appended sequence listing as SEQ ID NOS. 1 and 2, respectively.
The crystal structures of pectate lyases of Bacillus subtilis (1BN81 (and an R279K mutant, 2BSP2)), of Erwinia chrysanthemi (PelC: 2PEC3/1AIR4; PelC (R218K) in complex with substrate: ref 10; and PelE: 1PCL5), of Erwinia carotovora (PelC: 1PLU6), and of Bacillus sp. strain 2 KSM-P15 (1EE6) have been published. In addition, the crystal structures of the structurally very similar pectin lyases from Aspergillus niger (PlyA:1IDJ7/1IDK7 and PlyB:1QCX8) and of the polygalacturonase from Erwinia carotovora (1BHE9) are also known (1: R. Pickersgill, J. Jenkins, G. Harris, W. Nasser, J. Robert-Baudrouy; Nat.Struct.Biol. 1994, 1, 717;
2: R. Pickersgill, K. Worboys, M. Scott, N. Cummings, A. Cooper, J. Jenkins, D. Smith To Be Published;
3: M. D. Yoder, S. E. Lietzke, F. Jurnak; Structure, 1993, 1, 241;
4: M. D. Yoder, N. T. Keen, F. Jurnak; Science, 1993, 260, 1503;
5: M. D. Yoder, C. A. Dechaine, F. Jurnak; J. Biol.Chem. 1990, 265, 11429;
6: S. J. Tamaki, S. Gold, M. Robeson, S. Manulis, N. T. Keen; J Bacteriol. 1988, 170, 3468;
7: S. E. Lietzke, R. D. Scavetta, M. D. Yoder, F. A. Jurnak; Plant Physiol. 1996, 111, 73;
8: S. E. Lietzke, N. T. Keen, F. Jurnak; To Be Published;
9: Y. Kim, V. Mosser, N. Keen, F. Jurnak; J.Mol.Biol. 1989, 208, 365; N. T. Keen, S. Tamaki; J. Bacteriol. 1986, 168, 595;
10: M. D. Yoder, F. A. Jurnak; Plant Physiol. 1995, 107, 349;
11: O. Mayans, M. Scott, I. Connerton, T. Gravesen, J. Benen, J. Visser, R. Pickersgill, J. Jenkins; Structure, 1997, 5, 677;
12: J. Vitali, B. Schick, H. C. M. Kester, J. Visser, F. Jurnak; Plant Physiol. 1998, 116, 69;
13: R. Pickersgill, D. Smith, K. Worboys, J. Jenkins; .J Biol. Chem. 1998, 273, 24660;
14: R. D. Scavetta, S. R. Herron, A. T. Hotchkiss, N. Kita, N. T. Keen, J. A. E. Benen, H. C. M. Kester, J. Visser, F. Jurnak; Plant Cell 1999, 11, 1081;
15: M. Akita, A. Suzuki, T. Kobayashi,S. Ito, T. Yamane Crystallization And Preliminary X-Ray Analysis Of 2 High-Alkaline Pectate Lyase Acta Crystallogr., Sect.D V. 56 749 2000).
The crystal structures of a pectin methyl esterase (1QJV; Jenkins, J.; Mayans, O.; Smith, D.; Worboys, K.; Pickersgill, R. W. Journal of Molecular Biology, vol 305, 2001, 951-960) and a rhamnogalacturonase (1RMG; Petersen, T. N., Kauppinen, S., Larsen, S.: The crystal structure of rhamnogalacturonase A from Aspergillus aculeatus: a right-handed parallel beta helix. Structure 5 pp. 533 (1997)) have also been published.
Pectinases consist of an unusual backbone of parallel xcex2-strands coiled in a large right-handed helix. The parallel xcex2-helix contains three xcex2-strands pr. turn (PB1, PB2, and PB3) and consecutive turns stack one on to another to form a super-helix. Two of the xcex2-sheets form a xcex2-sandwich folded against each other in an anti-parallel manner. The third parallel xcex2-sheet is perpendicular to the xcex2-sandwich, resulting in an L-shaped cross-section. There is no direct sequence repetition in the xcex2-helix, however a typical characteristic of the domain is that the side-chains of residues at corresponding positions in consecutive xcex2-strands stack directly upon each other. The stacks can be of aliphatic (typically leucine, isoleucine and valine residues), hydrogen-bonded (typically asparagine residues, known as an asparagine ladder), or aromatic (typically tyrosine and phenylalanine residues) character. Stack amino acid side chains are found both within and outside the parallel xcex2-helix, forming a linear arrangement parallel to the axis of the xcex2 helix.
The three xcex2-sheets are separated by turns, termed T1 (between PB1 and PB2), T2 (between PB2 and PB3, introducing a 90xc2x0 change of backbone orientation), and T3 (between PB3 and PB1). It is within these regions that the largest diversity among the different pectinases is found, the most conserved regions being the xcex2-sheets PB2 and PB3 and the T2 turn. There is a large variety in the length of these turns, ranging from only two amino acids to tens of amino acids. The T3 turns are commonly lengthy and of more complex formation and constitute a loop region which together with the xcex2-helix (primarily PB1) confines the substrate binding crevice.
The N-terminal end of the parallel xcex2-helix domain is capped by an xcex1-helix that is structurally conserved although the sequence conservation is undetectable. The C-terminal end of the xcex2-helix is terminated by a loop structure with no specific conservation. The N-terminal tail packs against PB2 while the C-terminal tail lies across PB2 ending in a highly structurally (but not sequentially) conserved, amphipathic xcex1-helix, with the hydrophobic part packing against the T2 turn.
In the bottom of the pronounced substrate-binding cleft calcium binds to three aspartate residues, two of which are conserved for all pectate lyases and one that can also be glutamate. In addition, two arginines, one lysine and a proline all facing the substrate-binding cleft are conserved in the pectinase family. Mutation of the aspartates (one can be mutated to glutamate), the arginines or the lysine destroys the catalytic activity, however the exact catalytic mechanism is not fully understood.
A second cluster of invariant amino acids in the pectate lyases is located practically opposite to the Ca2+-binding site, that is, on the other side of the xcex2-heIix domain packing against the N-terminus. Even though this iWiDH region (SEQ ID NO: 27) is highly conserved throughout the pectinase family, the function of this is as yet unknown. It does not seem to be important for pectinolytic cleavage, but has been speculated to be involved in a second, yet unidentified, enzymatic function, or to be of importance in secretion of the enzyme always being of extra-cellular origin.
Hemicelluloses are the most complex group of non-starch polysaccharides in the plant cell wall. They consist of polymers of xylose, arabinose, galactose or mannose which are often highly branched and connected to other cell wall structures. Thus a multitude of enzymes is needed to degrade these structures (Ward and Young op.cit.). Xylanase, galactanase, arabinanase, lichenase and mannanase are some hemicellulose degrading enzymes.
Endo- and exo-xylanases and accessory enzymes such as glucuronidases, arabinofuranosidases, acetyl xylan esterase and ferulic acid or coumaric acid esterase have been summarized by Kormelink (1992, Ph.D.-thesis, University of Wageningen, The Netherlands). They are produced by a wide variety of micro-organisms and have varying temperature and pH optima.
Like other cell wall degrading enzymes (CWDE""S) galactanases occur in many micro-organisms (Dekker and Richards (1976), Adv. Carbohydrat. Chem. Biochem. 32, 278-319). In plant cell walls two types of arabinogalactans are present: type I 1,4 .beta.-galactans and type II 1,3/1,6 .beta.-galactans which have a branched backbone (Stephen (1983). In: The Polysaccharides. G. O. Aspinael (ed.). Ac. Press, New York, pp. 97-193). Both types of galactans require their own type of endo enzyme to be degraded. It can be expected that other enzymes, such as arabinan-degrading enzymes and exo-galactanases play a role in the degradation of arabinogalactans.
The hemicellulose 1,3-1,4-.beta.-glucan is a cell wall component present in cereal (barley, oat, wheat and rye) endosperm. The amount of .beta.-glucan in cereal endosperm varies between 0.7-8%. It is an unbranched polysaccharide built from cellotriose and cellotetraose residues linked by a 1,3-glucosidic bond. The ratio tri/tetra saccharose lies between 1.9 and 3.5.
Lichenase (EC 3.2.1.73) hydrolyse 1,4-beta-D-glucosidic linkages in beta-D-glucans containing 1,3- and 1,4-bonds. Lichenase reacts not on beta-D-glucans containing only 1,4-bonds such as for example in cellulose. Thus, damage of cellulose fibers in fabrics does not occur by the application of lichenase. Lichenases are produced by bacteria like B. amyloliguefaciens, B. circulans, B. licheniformis and plants (Bielecki S. et al. Crit. Rev. in Biotechn. 10(4), 1991, 275-304).
Arabinans consist of a main chain of .alpha.-L-arabinose subunits linked (.alpha.-(1- greater than 5) to another. Side chains are linked .alpha.-(1- greater than 3) or sometimes .alpha.-(1- greater than 2) to the main alpha.-(1- greater than 5)-L-arabinan backbone. In apple, for example, one third of the total arabinose is present in the side chains. The molecular weight of arabinan is normally about 15 kDa.
Arabinan-degrading enzymes are known to be produced by a variety of plants and micro-organisms. Three enzymes obtainable from A.niger have been cloned by molecular biological techniques (EP-A-506190). Also arabinosidase from bacteria such as Bacteroides has been cloned (Whitehead and Hespell (1990). J. Bacteriol. 172, 2408).
Galactomannans are storage polysaccharides found in the seeds of Leguminosae. Galactomannans have a linear (1xe2x86x924)-.beta.-mannan backbone and are substituted with single (1xe2x86x926).alpha.-galactose residues. For example in guar gum the ratio mannose/galactose is about 2 to 1. Galactomannans are applied as thickeners in food products like dressings and soups.
Mannanase enzymes are described in PCT application WO 93/24622.
Glucomannan consists of a main chain of glucose and mannose. The main chain may be substituted with galactose and acetyl groups; mannanases can be produced by a number of microorganisms, including bacteria and fungi.
To summarise, it can be said that a large number of plant cell wall degrading enzymes exist, produced by different organisms. Depending on their source the enzymes differ in substrate specificity, pH and temperature optima, Vmax, Km etc. The complexity of the enzymes reflects the complex nature of plant cell walls, which differ strongly between plant species and within species between plant tissues.
It is an object of the present invention to provide a cell-wall degrading enzyme variant, especially a pectin degrading enzyme variant, which exhibits improved performance over the known microbial cell-wall degrading enzymes when applied e.g. in detergents or in textile industry processes.
The inventors have now found that certain amino acid substitutions in cell-wall degrading enzymes having a structure including a xcex2-helix result in enzyme variants having improved performance in the neutral or alkaline pH range, especially improved thermostability when determined by DSC (Disc Scanning Calorimetry) or by a Pad-Steam application test.
In a preferred embodiment of the invention, variants of the Bacillus licheniformis pectate lyase (EC 4.2.2.2) encoded by SEQ ID NO: 1 exhibit improved properties over the parent pectate lyase, the improved properties being advantageous when the enzyme is applied industrially. The inventors have provided such variants by having succeeded in identifying certain positions in the protein sequence in which positions the naturally occurring amino acid residue may be substituted or deleted or in which positions one or more amino acid residues may be inserted with the purpose of providing an improved pectate lyase variant, and have further provided a method of constructing cell-wall degrading enzyme variants with improved performance in industrial applications.
Accordingly, in a first aspect the present invention relates to a variant of a cell-wall degrading enzyme having a beta-helix structure, which variant holds at least one substituent in a position determined by (i) identifying all residues potentially belonging to a stack; (ii) characterising the stack as interior or exterior; (iii) characterising the stack as polar (typically asparagine, serine, threonine) or hydrophobic (either aliphatic: leucine, isoleucine or valine; or aromatic/heteroaromatic: phenylalanine, tyrosine, histidine, and less often tryptophan) based on the dominating characteristics of the parent or wild-type enzyme stack residues and/or its orientation relative to the beta-helix (interior or exterior); (iv) optimising all stack positions of a stack either to hydrophobic aliphatic amino acids, hydrophobic aromatic/heteroaromatic amino acids (preferably histidine alone, tyrosine and phenylalanine alone or in combination) or polar amino acids (preferably asparagine) by allowing mutations within one or all positions to amino acids belonging to one of these groups; (v) measuring thermostability of the variants by DSC or an application-related assay such as a Pad-Steam application test; and (vi) selecting the stabilized variants. Alternatively, the variants may be provided by scanning the X-ray structure for positions that may be mutated into a proline residue; and mutating at least one of these positions into a proline; or by scanning the x-ray structure for positions that may be mutated into cysteine residues in order for these to form disulfide bridges and thereby stabilize the structure; and mutate at least one of these positions into a cysteine; or by initiating molecular dynamics calculations specifying different temperatures using the x-ray structure.
In a preferred embodiment, the invention relates to a variant of a wild-type parent pectate lyase (EC 4.2.2.2) having the conserved amino acid residues D111, D141 or E141, D145, K165, R194 and R199, optionally also W123, D125 and H126, when aligned with the pectate lyase comprising the amino acid sequence of SEQ ID NO: 2, in which the variant is substituted in at least one position selected from the group consisting of the positions 41, 55, 71, 72, 82, 83, 90, 100, 102, 114, 129, 133, 136, 144, 160, 163, 167, 168, 169, 189, 192, 197, 198, 200, 203, 207, 220, 222, 230, 232, 236, 237, 238, 244, 246, 261, 262, 265, 269, 282, 283, 284, 285, 288 and 289. It is believed that the novel enzyme will be classified according to the Enzyme Nomenclature in the Enzyme Class EC 4.2.2.2. However, it should be noted that it is contemplated that the pectate lyase variant of the invention also exhibits catalytic activity on pectin (which may be esterified) besides the activity on pectate and polygalacturonides conventionally attributed to enzymes belonging to EC 4.2.2.2.
Within another aspect, the present invention provides an isolated polynucleotide molecule prepared from the DNA molecule comprising the DNA sequence of SEQ ID NO: 1 by conventional methods such as site-directed mutagenesis.
Within yet another aspect of the invention there is provided an expression vector comprising the following operably linked elements: (a) a transcription promoter, (b) the polynucleotide molecule of the invention, (c) degenerate nucleotide sequences of (a) or (b); and a transcription terminator.
Within yet another aspect of the present invention there is provided a cultured cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses the polypeptide encoded by the DNA segment.
Within another aspect of the present invention there is provided an enzyme composition comprising the pectate lyase variant of the invention in combination with other enzymes.
Within another aspect of the present invention there are provided methods for producing a polypeptide according to the invention comprising culturing a cell into which has been introduced an expression vector as disclosed above, whereby said cell expresses a polypeptide encoded by the DNA segment and recovering the polypeptide.
In comparison with the wild-type cell-wall degrading enzyme, especially a wild-type pectate lyase, it is contemplated that the variant of the invention exhibits increased thermal stability, either due to further stabilization of the xcex2-helix structure of the protein by amino acid substitution in positions within the aliphatic and aromatic stacks of amino acid side chains, or to further stabilization of the binding cleft or the C-terminal turn. Increased thermostability of an enzyme is indeed very useful in many industrial applications which advantageously can be carried out at a temperature above the temperature optimum for the enzymatic activity of the wild-type enzyme.
The cell-wall degrading enzyme variant of the invention is useful for the treatment of cellulosic material, especially cellulose-containing fiber, yarn, woven or non-woven fabric, treatment of mechanical paper-making pulps or recycled waste paper, and for retting of fibres. The treatment can be carried out during the processing of cellulosic material into a material ready for garment manufacture or fabric manufacture, e.g. in the desizing or scouring step; or during industrial or household laundering of such fabric or garment.
Accordingly, in further aspects the present invention relates to a detergent composition comprising an enzyme variant having substantial cell-wall degrading activity; and to use of the enzyme variant of the invention for the treatment of cellulose-containing fibers, yarn, woven or non-woven fabric.
The enzyme variant of the invention, especially the pectate lyase variant, is very effective for use in an enzymatic scouring process in the preparation of cellulosic material e.g. for proper response in subsequent dyeing operations.